Inhibiting protein-protein binding interactions with designed molecules is a frontier challenge in biomedical science. Protein function is a direct result of 3-dimensional folded conformation. Thus, creating a molecule that mimics the function of a particular protein requires that the molecule manifest key structural features of the folded state of the prototype. Significant advances have been made in the mimicry of simple secondary structures; however, there is an unmet need for a design strategy capable of generating unnatural species that show the same complex tertiary folds as natural proteins. A long-term goal of research in the PI's lab is to invent a suite of tools for the design of natural protein analogues that manifest key structural and functional features of the prototype on scaffolds with enhanced folded and/or physiological stability. The overall objective of the present proposal is to develop a sequence-based method for the mimicry of protein tertiary folding by unnatural backbones and to apply this method to two biomedically significant targets where a tertiary fold is essential for function. The central hypothesis guiding the work is that any natural protein sequence can serve as the starting point for the design of its own unnatural analogue, provided systematic rules for backbone modification exist. The rationale motivating this work is that a general method for mimicry of protein tertiary folding by unnatural backbones that is truly sequence-based will open the possibility to address folds of arbitrary complexity. The central hypothesis will be tested through pursuit of three specific aims: (1) Establish design principles for sequence-based mimicry of protein tertiary folding by unnatural-backbone oligomers; (2) Develop a general method for the conversion of phage-derived affinity ligands to protease-resistant analogues and apply this method to create tumor imaging agents; (3) Design inhibitors of a conserved protein-protein interaction common to disease progression in malaria and toxoplasmosis. Expected outcomes of the proposed work include a general strategy for the design of protease-resistant species that manifest tertiary folding patterns of natural proteins, a paradigm to connect phage-based sequence selection to unnatural backbones, tumor imaging agents that recognize cancer-associated cell-surface proteins, and oligomers that target a common pathway in infection by malaria and other parasites. The significance of the research stems from the wealth of new functions that will become possible when complex tertiary folding patterns are accessible to unnatural backbones. The innovation of the proposed idea arises from addressing the important challenge of tertiary structure mimicry by a design approach that is sequence-based and generalizable to proteins beyond those discussed in the present proposal.